Cholesterol is the major steroid constituent of animal tissue, and an essential component of plasma and cell membranes. Cholesterol is a 3-hydroxy sterol having a perhydro-1,2-cyclopenenophenanthrene ring system and an aliphatic side chain at position 17. Because it is insoluble in body fluids, cholesterol must be transported through the bloodstream by carriers such as low-density lipoprotein (LDL).
Cholesterol is an extremely important biological molecule that has roles in membrane structure as well as being a precursor for the synthesis of the steroid hormones and bile acids.
The synthesis and utilization of cholesterol must be tightly regulated in order to prevent over-accumulation and abnormal deposition within the body. Of particular importance clinically is the abnormal deposition of cholesterol and cholesterol-rich lipoproteins in the coronary arteries. Such deposition, eventually leading to atherosclerosis, is the leading contributory factor in diseases of the coronary arteries.
A portion of the cholesterol in the body derives from biosynthesis de novo. In the process of generating cholesterol, lanosterol is the first sterol formed and conversion to cholesterol requires at least a dozen additional steps.
Through a series of reactions, lanosterol is converted to cholesterol (FIG. 1). The conversion of lanosterol, through zymosterol, and desmosterol, including several intermediates is depicted in FIG. 2.
Normal healthy adults synthesize cholesterol at a rate of approximately 1 g/day and consume approximately 0.3 g/day. A relatively constant level of cholesterol in the body (150-200 mg/dL) is maintained primarily by controlling the level of de novo synthesis. The level of cholesterol synthesis is regulated in part by the dietary intake of cholesterol. Cholesterol from both diet and synthesis is utilized in the formation of membranes and in the synthesis of the steroid hormones and bile acids (see below). The greatest proportion of cholesterol is used in bile acid synthesis.
Excessive accumulation of cholesterol has been implicated as the primary causative factor in a number of diseases, including atherosclerosis, which is characterized by an abnormal hardening and thickening of the arterial walls due to the accumulation and deposition of fatty materials, including cholesterol. This can lead to thrombosis and infarction.
The cholesterol metabolite 26-hydroxycholesterol, now known as 27-hydroxycholesterol, has been previously shown to be associated with cholesterol synthesis (U.S. Pat. No. 4,427,668). More specifically, a reduced level of 27-hydroxycholesterol in the serum was found to be associated with cholesterol build up in the tissues; thus, the administration of 27-hydroxycholesterol was proposed as a method for reducing the rate of cholesterol synthesis in the body. Thereafter, as disclosed in U.S. Pat. No. 4,939,134, it was discovered that 27-amino cholesterol and certain amino-substituted analogs and derivates were more potent inhibitors of cholesterol synthesis and accumulation than 27-hydroxycholesterol. 27-hydroxycholesterol was later found to be effective in reducing the occurrence of restenosis following injury to the blood vessels lumen (U.S. Pat. No. 5,376,652). Individuals with a genetic defect in producing 27-hydroxycholesterol exhibit accelerated atherosclerosis and die early in life of severe coronary artery disease. The molecular basis of this genetic disease is a mutation in the CYP 27 gene, which results in a lack of cholesterol 27-hydroxylase activity.
The term “oxysterol”, although not precisely defined, refers generically to derivatives of cholesterol having one, or occasionally more, hydroxyl or oxo groups, in addition to the 3β-OH group of cholesterol (Russell, D. W. (2000) Biochim Biophys Acta. 1529:126-135; Javitt, N. B. (2000) Biochim Biophys Acta. 1529:136-141). For the most part these “oxysterols”, such as 27-hydroxycholesterol and 24s-hydroxycholesterol, are endogenously-derived derivatives of cholesterol. Others such as 25-hydroxycholesterol, which can be generated endogenously (Lund, E. G. et al. (1998) J. Biol. Chem. 273:34316-34327), are also well-known oxidation products.
Kandutsch et al. reported that oxygenated cholesterol has an inhibitory effect on the proliferation of fibroblasts and lymphocytes in vitro, perhaps by inhibiting hydroxymethylglutaryl coenzyme A reductase (HMG CO-A reductase), the rate-limiting enzyme in cholesterol biosynthesis (Kandutsch et al. (1978) Science 201:498). The inhibitory effect by oxysterols on vascular smooth muscle cells has been suggested to be a toxic effect (Zhou et al. (1993) Proc. Soc. Exp. Biol. Med. 202: 75-80).
It is now well established that one of the basic mechanisms for regulating gene expression is via the binding of “oxysterols” to nuclear receptors that in turn modulate gene expression by affecting their promoter region (Lu, T. T. et al. (2001) J. Biol. Chem. 276:37735-37738; Lobaccaro, J. M. et al. (2001) Ann Endocrinol (Paris) 62:329-247). These nuclear receptors are often referred to as “orphan” nuclear receptors, with the term “orphan” referring to their status before known oxysterols were identified that bind to these receptors to modulate both their expression and the genes with appropriate target promoter regions. The oxysterols 24,25-epoxy cholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol are now known to function as ligands for nuclear receptors that modulate other determinants of cholesterol homeostasis (Janowski et al. (1996) Nature 383:728-31). The oxysterol 25R,26-hydrocholesterol (also referred to as 27-hydroxycholesterol) appears to have a role in preventing cholesterol accumulation in arteries. Although oxysterols can function as ligands for nuclear receptors, they can also have biological activities unrelated to this function. Bramlett et al. recently discussed a natural product ligand of the oxysterol receptor LXR, the fungal metabolite paxilline (Bramlett, K. S. et al. (2003) J. Pharmcol Exp. Therap 307:291-296).
Therefore, in view of the fact that macronutrients and metabolites, including endogenous sterols, affect gene expression through nuclear receptors as well as directly influence metabolic processes, there exists a need in the art for novel ligands for these receptors and modulators of these processes. Furthermore, there exists a need for additional approaches to controlling and modulating cholesterol levels and endogenous sterol constituents.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.